This application is directed to inhibitors of nitric oxide synthase, and in particular cyclic amidines.
Nitric Oxide in Biology
The emergence of nitric oxide (NO), a reactive, inorganic radical gas as a molecule contributing to important physiological and pathological processes is one of the major biological revelations of recent times. This molecule is produced under a variety of physiological and pathological conditions by cells mediating vital biological functions. Examples include endothelial cells lining the blood vessels; nitric oxide derived from these cells relaxes smooth muscle and regulates blood pressure and has significant effects on the function of circulating blood cells such as platelets and neutrophils as well as on smooth muscle, both of the blood vessels and also of other organs such as the airways. In the brain and elsewhere nitric oxide serves as a neurotransmitter in non-adrenergic non-cholinergic neurons. In these instances nitric oxide appears to be produced in small amounts on an intermittent basis in response to various endogenous molecular signals. In the immune system nitric oxide can be synthesized in much larger amounts on a protracted basis. Its production is induced by exogenous or endogenous inflammatory stimuli, notably endotoxin and cytokines elaborated by cells of the host defense system in response to infectious and inflammatory stimuli. This induced production results in prolonged nitric oxide release which contributes both to host defense processes such as the killing of bacteria and viruses as well as pathology associated with acute and chronic inflammation in a wide variety of diseases. The discovery that nitric oxide production is mediated by a unique series of three closely related enzymes, named nitric oxide synthases, which utilize the amino acid arginine and molecular oxygen as co-substrates has provided an understanding of the biochemistry of this molecule and provides distinct pharmacological targets for the inhibition of the synthesis of this mediator, which should provide significant beneficial effects in a wide variety of diseases.
Nitric Oxide Synthases
Nitric oxide and L-citrulline are formed from L-arginine via the dioxygenase activity of specific nitric oxide synthases (NOSs) in mammalian cells. In this reaction, L-arginine, O.sub.2 and NADPH are cosubstrates while FMN, FAD and tetrahydrobiopterin are cofactors. NOSs fall into two distinct classes, constitutive NOS (cNOS) and inducible NOS (iNOS). Two constitutive NOSs have been identified. They are:
(i) a constitutive, Ca.sup.++ /calmodulin dependent enzyme, located in the endothelium and elsewhere (ecNOS or NOS 3), that releases NO in response to receptor or physical stimulation, PA1 (ii) a constitutive, Ca.sup.++ /calmodulin dependent enzyme, located in the brain (ncNOS or NOS 1) and elsewhere, that releases NO in response to receptor or physical stimulation, PA1 (iii) a Ca.sup.++ independent enzyme which is induced after activation of vascular smooth muscle, macrophages, endothelial cells, and a large number of other cells by endotoxin and cytokines. Once expressed, this inducible NO synthase produces NO in relatively large amounts for long periods of time. PA1 X is selected from O, S(O).sub.m, NH, and NR.sub.6, PA1 m is 0, 1 or 2; PA1 R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are each independently selected from the group consisting of PA1 or when two members of the group R.sub.1, R.sub.2, R.sub.3 and R.sub.4 including the optional substituents present thereon reside on the same carbon atom of Formula I, or two of the group R.sub.1, R.sub.2, R.sub.3 and R.sub.4 including the optional substituents present thereon reside on adjacent atoms of Formula I, said two members may optionally be joined, such that together with the carbon atom to which they are attached there is formed a saturated or unsaturated monocyclic ring of 5, 6 or 7 atoms, said monocyclic ring optionally containing up to three hetero atoms selected from N, O or S, PA1 or when a member of the group R.sub.1, R.sub.2, R.sub.3 and R.sub.4 including the optional substituents present thereon resides on an atom adjacent to the N on which R.sub.6 resides, said member may optionally be joined with R.sub.6, such that together with the N on which R.sub.6 resides and the carbon on which said member resides there is formed a saturated or unsaturated monocyclic heterocycle of 5, 6 or 7 atoms, said monocycle optionally containing up to three hetero atoms selected from N, O or S, PA1 R.sub.5 is selected from the group consisting of PA1 R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are each independently selected from the group consisting of PA1 R.sub.5 is selected from the group consisting of PA1 R.sub.5 is selected from the group consisting of PA1 R.sub.2 is hydrogen or methyl; PA1 R.sub.4 is hydrogen or methyl; PA1 R.sub.1 and R.sub.3 are each independently selected from PA1 R.sub.5 is hydrogen.
The third isoform identified is inducible NOS (iNOS or NOS 2):
Spectral studies of both the mouse macrophage iNOS and rat brain ncNOS have shown that these enzymes (which have been classified as P-450-like enzymes from their CO-difference spectra) contain a heme moiety. The structural similarity between NOS and the P-450/flavoprotein complex suggests that the NOS reaction mechanism may be similar to P-450 hydroxylation and/or peroxidation. This indicates that NOS belongs to a class of flavohemeproteins which contain both heme and flavin binding regions within a single protein in contrast to the multiprotein NADPH oxidase or Cytochrome P-450/NADPH Cyt c reductase complexes.
Distinct Functions of NO Produced by Different Nitric Oxide Synthases.
The NO released by the constitutive enzymes (NOS 1 and NOS 3) acts as an autocoid mediating a number of physiological responses. Two distinct cDNAs accounting for the activity of NOS 1 and NOS 3 in man have been cloned, one for NOS 1 (Nakane et. al., FEBS Letters, 316, 175-182, 1993) which is present in the brain and a number of peripheral tissues, the other for an enzyme present in endothelium (NOS 3) (Marsden et. al., FEBS Letters, 307, 287-293, 1992). This latter enzyme is critical for production of NO to maintain vasorelaxation. A second class of enzyme, iNOS or NOS 2, has been cloned from human liver (Geller et. al., PNAS, 90, 3491-5, 1993), and identified in more than a dozen other cells and tissues, including smooth muscle cells, chondrocytes, the kidney and airways. As with its counterpart from the murine macrophage, this enzyme is induced upon exposure to cytokines such as gamma interferon (IFN-.gamma.), interleukin-1.beta. (IL-1.beta.), tumor necrosis factor (TNF-.alpha.) and LPS (lipopolysaccharide). Once induced, iNOS expression continues over a prolonged period of time. The enzyme does not require exogenous calmodulin for activity.
Endothelium derived relaxation factor (EDRF) has been shown to be produced by NOS 3 (Moncada et. al., Pharmacol. Reviews, 43, 109-142, 1991). Studies with substrate analog inhibitors of NOS have shown a role for NO in regulating blood pressure in animals and blood flow in man, a function attributed to NOS 3. A transgenic mouse deficient in functional NOS 3 was shown to be hypertensive, thus validating the role of NO synthesis by NOS 3 in the regulation of blood pressure (Huang et al., Nature, 377, 239-242, 1995). NO has also been shown to be an effector of the cytotoxic effects of activated macrophages (Nathan, FASEB J., 6, 3051-64, 1992) for fighting tumour cells and invading microorganisms (Wright et al., Card. Res., 26, 48-57, 1992 and Moncada et al., Pharmacological Review, 43, 109-142, 1991). It also appears that the adverse effects of excess NO production, in particular pathological vasodilation and tissue damage, may result largely from the effects of NO synthesized by the NOS 2.
NO generated by NOS 2 has been implicated in the pathogenesis of inflammatory diseases. In experimental animals hypotension induced by LPS or TNF-.alpha. can be reversed by NOS inhibitors and reinitiated by L-arginine (Kilbourn et. al., PNAS, 87, 3629-32, 1990). Conditions which lead to cytokine-induced hypotension include septic shock, hemodialysis (Beasley and Brenner, Kidney Int., 42, Suppl., 38, S96-S100, 1992) and IL-2 therapy in cancer patients (Hibbs et. al., J. Clin. Invest., 89, 867-77, 1992). NOS 2 is implicated in these responses, and thus the possibility exists that a NOS inhibitor would be effective in ameliorating cytokine-induced hypotension. Recent studies in animal models have suggested a role for NO in the pathogenesis of inflammation and pain and NOS inhibitors have been shown to have beneficial effects on some aspects of the inflammation and tissue changes seen in models of inflammatory bowel disease, (Miller et. al., J. Pharmacol. Exp. Ther., 264, 11-16, 1990) and cerebral ischemia and arthritis (Ialenti et. al., Br. J. Pharmacol., 110, 701-6, 1993; Stevanovic-Racic et al., Arth. & Rheum., 37, 1062-9, 1994). Moreover transgenic mice deficient in NOS 1 show diminished cerebral ischemia (Huang et. al., Science, 265, 1883-5, 1994) and transgenic mice deficient in NOS 2 exhibit enhanced survivability in a model of LPS-induced shock (MacMicking et al. Cell 81, 641-650, 1995) and Wei et al. Nature 375, 408-411, 1995)).
Further conditions where there is an advantage in inhibiting NO production from L-arginine include therapy with cytokines such as TNF, IL-1 and IL-2 or therapy with cytokine-inducing agents, for example 5,6-dimethylxanthenone acetic acid, and as an adjuvant to short term immunosuppression in transplant therapy. In addition, compounds which inhibit NO synthesis may be of use in reducing the NO concentration in patients suffering from inflammatory conditions in which an excess of NO contributes to the pathophysiology of the condition, for example adult respiratory distress syndrome (ARDS) and myocarditis.
There is also evidence that an NO synthase enzyme may be involved in the degeneration of cartilage which takes place in autoimmune and/or inflammatory conditions such as arthritis, rheumatoid arthritis, chronic bowel disease and systemic lupus erythematosis (SLE). It is also thought that an NO synthase enzyme may be involved in insulin-dependent diabetes mellitus. Therefore, a yet further aspect of the present invention provides cyclic amidine derivatives or salts thereof in the manufacture of a medicament for use in cytokine or cytokine-inducing therapy, as an adjuvant to short term immunosuppression in transplant therapy, for the treatment of patients suffering from inflammatory conditions in which an excess of NO contributes to the pathophysiology of the condition.